Lysotracker red dye

Cells derived from patients with Niemann Pick disease Type C (NPC) exhibit the NPC disease phenotype of enlarged lysosome due to cholesterol accumulation in lysosomes that can be measured by the LysoTracker dye staining assay in a fluorescence imaging detection mode or in a fluorescence intensity mode by a plate reader.^{10 (link), 15 (link), 16 (link)} NSCs were plated at 1000 cells/well in 5µl of complete StemPro NSC Serum Free Medium supplemented with 10µg/ml rhVTN-N in 1536-well, black, clear bottom assay plates and incubated 4 hours at 37 °C and 5% CO₂. Columns 1 and 2 were used as the negative control with the wild type NSCs and columns 3 through 48 were NPC NSCs. Methyl-β-cyclodextrin (MΒCD) and hydroxypropyl-β-cyclodextrin (HPBCD) which reduce cholesterol accumulation in NPC cells and thus decrease the enlarged lysosomes in NPC cells were added to the assay plate at 23nL/well as positive controls. After a 96 hours incubation, 200 nM Lysotracker-red dye (Life Technologies) was added to the assay plates to stain lysosomes. The fluorescence intensity (E_x = 570 nm, E_m = 590 nm) was measured with a bottom read mode in the Tecan plate reader. The data from the homogenous LysoTracker staining assay was normalized using the WT NSCs as a negative control and DMSO-treated NPC NSCs as a positive control to calculate IC₅₀ values, S/B ratio, CV, and Z-factor.

The analysis of lysosomal membrane permeability was performed using LysoTracker™ red dye purchased from Invitrogen (CAT# L7528) following the manufacturer’s protocol. In brief, SH-SY5Y cells were grown in a 96-well plate and were transfected and treated as above. Following treatment, cells were incubated with 100 nM LysoTracker™ dye for 30 min. Post-incubation, cells were washed with PBS multiple times and were analyzed using a SpectraMax fluorescence plate reader at excitation/emission of 445 nm/505 nm. A decrease in fluorescence correlates with reduced number of lysosomes.